Composition and method for determining uric acid



United States Patent C) Indiana No Drawing. Filed Dec. 14, 1964, Ser. No. 418,333 18 Claims. (Cl. 195-103.5)

This invention relates to a new and improved composition for catalyzingenzymatic oxidation, to a process therefor and to a method for the determination of purines. In one of its more particular aspects it relates to an improved test for the qualitative and quantitative determination of uric acid in body fluids.

In human metabolism, there is a constant endogenous conversion of ingested nucleoproteins to purines and pyridimines such as adenine, guanine, thymine, cytosine and uracil. The purines, by a catabolic process, then undergo further deamination and partial oxidation to uric acid, which, in man, is normally excreted in the urine. Such a metabolic process results in a nominal concentration of uric acid in the blood and urine of man at all times.

Ingestion of purine-containing food normally has no effect on the uric acid blood serum content, except in the case of renal insufficiency, in which event the concentration is elevated. In certain other pathological conditions not associated with dietary ingestion of purinecontaining food, for example, uremia and gout, there is an abnormal increase in the amount. of uric acid found in the blood serum. It is also known that the concentration of uric acid in the blood serum is raised in conditions associated with excessive destruction of the nuclei of white blood corpuscles, for example, leukemia and pneumonia.

Medical science has long recognized the usefulness of a test for uric acid in the blood serum as an aid in diagnosing the foregoing conditions, and in some instances, distinguishing between closely related abnormal condi tions, for example, gout and arthritis. Gout is characterized by an abnormal increase in blood serum uric acid whereas arthritis does not exhibit such increase. It is therefore considered extremely desirable to provide a test which is simple and economical and yet affords a precise and specific determination of the concentration of uric acid in blood serum.

Uric acid is normally found in blood serum in varying small quantities, that is, from about 0.7 to about 6.0 milligrams per 100 ml. of blood serum and is generally reported as milligram percent (mg. percent). In the abnormal conditions enumerated above, the uric acid content in the blood serum often attains values of mg. percent or higher.

The prior art has disclosed a number of methods for determining uric acid in blood serum. Among the more widely used conventional methods are colorimetric procedures utilizing blood filtrates. Some of these procedures depend upon the precipitation of uric acid from the blood filtrate, for example, as a silver salt, and the formation therewith of a chromogenic adduct by reaction with either a phosphotungstate or arsenotungstate. Other methods utilizing the blood filtrate depend upon the direct treatment of the filtrate with a tungstic acid in the presence of a cyanide-urea solution to develop a color which 3,335,069 Patented Aug. 8, 1967 is then measured using the techniques of those skilled in the art to give a quantitative estimate of uric acid concentration.

More recently methods have been proposed which involve the catalyzed oxidation of uric acid to allantoin and hydrogen peroxide. This oxidation is generally accomplished in the presence of atmospheric oxygen and utilizes a material having uricase activity. In such methods a spectrophotometer may be employed to measure the disappearance of the characteristic uric acid spectrum during its conversion to allantoin and hydrogen peroxide. Another method utilizes a colorimetric means for measuring the hydrogen peroxide that is produced in stoichiometric amounts during such degradation of uric acid.

In the enzymatic conversion test, where the amount of hydrogen peroxide is produced is directly proportional to the amount of uric acid present in the blood serum, the hydrogen peroxide is detected by means of a color change produced upon oxidation of a color forming substance in the presence of a substance having peroxidative activity. The color obtained is then compared visually to standards, or measured electronically, which gives a quantitative estimation of uric acid present in the fluid being tested.

While this enzyme conversion test is an effective analytical method for measuring the quantity of uric acid in fluids, such test has one major disadvantage in that it requires the use of a specially purified uricase to efiect a stoichiometric production of hydrogen peroxide. If a less pure or, as used herein, a bulk enzyme is utilized in the above analytical scheme, the color development, which is dependent upon the hydrogen peroxide produced, is not directly proportional to the uric acid present. Although it has been found that reaction inhibition occurs, in some degree, in most impure or bulk enzymes which effect the oxidation of purine base compounds, this inhibitory action is found particularly in uricase. It is believed that such inhibition may be due to impurities present in the bulk uricase. In many instances purification techniques must be repeated several times before an effective uricase is obtained. This extensive purification process may increase the cost of the purified enzyme from 10 to 50 times that of the impure or bulk enzyme.

It is an object of this invention to provide an improved enzyme containing composition which catalyzes the oxidation of purinesi It is another object of this invention to provide an improved means for detecting purines and particularly uric acid in blood serum.

Other objects will become apparent from the ensuing disclosure and appended claims.

It has been unexpectedly found that in the enzymatic conversion test for purines such as uric acid, a directly proportional conversion of the uric acid to hydrogen peroxide and allantoin is obtained, along with excellent reproducibility, if a heterocyclic nitrogen containing comenzyme were not used, the peroxides produced in the enzymatic reaction did not accurately represent the concentration of uric acid present ina solution under test. Although it is not clearly understood how the substituted quinoline compound effects a stoichiometric conversion, it is believed that the nitrogen containing heterocyclic compound in some manner complexes the impurities present in the bulk enzyme which otherwise cause peroxide decomposition or inactivity.

Specifically, the test composition of this invention comprises in combination an enzyme reaction system and a peroxide detecting system.

In the preferred embodiment of the present invention, the enzyme reaction system comprises a bulk enzyme which exhibits uricase activity, a buffer to maintain the reaction system at its optimal pH and a substituted quinoline compound having the general formula:

wherein R and R each independently represents a hydrogen atom or a lower alkyl, lower alkoxy, or formyl group and R represents a hydrogen atom or lower alkyl, lower alkoxy, carboxyl or formyl group. Preferably, the lower alkyl and lower alkoxy groups contain less than four carbon atoms.

A mono-substituted or di-substituted quinoline compound wherein position 6 is one of the positions substituted, is generally the preferred additive for most enzymes and particularly where the enzeme is uricase.

Examples of some of the more effective mono-substituted and di-substituted quinoline compounds include the methoxy substituted quinolines such as G-methoxyquinoline, 4-methoxyquinoline, 6-methoxy-2-methylquinoline; methyl substituted quinoline compounds such as 4,6- dimethylquinoline, 6-methylquinoline, 2,6-dimethylquinoline and the like; c-arboxyl substituted quinoline compounds such as 6-quinoline carboxylic acid, 2-methyl-6- quinoline carboxylic acid and the like; and formyl substituted quinoline compounds such as 6-aldehydequinoline, 4-aldehydequinoline and the like.

The amount of the substituted quinoline compound which is required to effectively protect against peroxide decomposition or to otherwise provide for a proportional conversion of the purine depends largely on the purity of the enzyme used. If the starting material is relatively pure to begin with, a lesser quantity of the substituted quinoline compound is generally required. On the other hand, if the bulk enzyme is relatively impure, then a higher concentration of the substituted quinoline compound is required. The minimum concentration of the substituted quinoline compound that can be used for a particular type of enzyme is determined experimentally with little difficulty. For example, when 1 gram of a bulk or impure enzyme derived from hog liver is used, generally 0.2 to 2 grams of 6-methoxyquinoline compound is used. However, with other types and grades of uricase more or less of the substituted quinoline compound may be required.

When uricase is utilized as the enzyme it is desirable to employ a buffer which is effective to maintain the composition within a pH range of from about 8.5 to about 10.0 with a preferable pH of from about 9.0 to 9.2 being maintained. Buffers which may be used include, for example, borate buffer, glycine and tris(hydroxymethyl)- aminomethane. The concentration of the buffer is not critical. However, it is preferable to use a relatively dilute bufl er solution and for this purpose a 0.1 M solution is recommended.

If the fluid being tested is blood serum, such serum contains the enzyme catalase. Catalase destroys hydrogen peroxide and it is therefore necessary to include in the formulation of the enzyme reaction system a catalase inhibiting substance. For this purpose, sodium azide has been found to be efiective.

In many instances, it has been found desirable to include with the enzyme reaction system a stabilizing agent for the uricase. Since uricase in dilute solution is sensitive to traces of metallic ions, it is desirable to stabilize the uricase by utilizing any standard chelating agent such as ethylenediamine, 1,2-propylenediamine or other diamines or substituted diamines which are effective to prevent inhibition of uricase by bivalent or multivalent ions. A preferred stabilizing agent, for example, is ethylenediaminetetraacetic acid.

The enzyme reaction system comprising the impure enzyme and the substituted quinoline may be utilized in various ways. For example, the ingredients may be mixed in liquid form or, depending upon the availability of solid enzymes, in powder form. The liquid formulation may then be lyophilized to give a formulation which can be readily reconstituted for later use simply by the addition of water thereto. The powder may also be tabletted, encapsulated or pelletized for convenience in use.

The hydrogen peroxide detecting system which is used to indicate and, if desired, quantitate the peroxide produced in the uricase catalyzed oxidation of uric acid, comprises a chromogen, a substance having peroxidative activity and a buffer to provide a pH at which the chromogen oxidation may be elfectively carried out. In buffering the peroxide detecting system, it is desirable to provide a pH range of from about 3.7 to about 6.8, with a preferable range of from about 4.5 to 5.5. Butters which may be used include, for example, citrate, succinate and tris(hydroxymethyl)methylamine rnalonate. One reason for requiring a pH within the acid range is to insure that the most stable color is developed, since it has been observed that at a higher pH the color fades rapidly.

Various chromogens may be used to provide the measure of the quantity of peroxide produced by the enzyme reaction system. Particularly, derivatives of aniline and phenol such as, for example, orthotolidine, orthotoluidine, p-toluidine, o-phenylenediamine, N,N-dimethyl-pphenylenediamine, benzidine, o-anisidine, p-anisidine, dianisidine, o-c'resol, m-cresol, wnaphthol, B-naphthol, catechol, guaiacol, pyrogallol, 2,7-diaminofluorene, leucoindophenols, and gum guaiac can be employed.

The enzyme peroxidase is one of a group of materials known to have peroxidative activity, that is, having the capacity to catalyze the oxidation of chromogenic materials with hydrogen peroxide. Peroxidase may obtained from horseradish, fig leaves or potatoes. Other materials known to have peroxidative activity, as defined above, include, for example, normal whole blood, red blood cells, lyophilized whole blood and similar rnetalloporphyrin materials, both natural and synthetic. A mixture of potassium iodide and sodium molybdate as well as other Water soluble iodides such as sodium iodide and ammonium iodide and other soluble molybdates such as potassium molybdate, sodium molybdate and ammonium molybdate have been found useful for catalyzing the color reaction. Such compounds may also be used as color forming substances, either alone or in combination with the chromogenic compounds described above. Urohemin and other porphyrin substances known to be effective for catalyzing oxidation reactions may also be used. In the case of the metalloporphyrins, although hemin is preferred, various complex forming compounds which activate certain other metalloporphyrins not operable per se can be used in combination with such other metalloporphyrins to produce an effective catalytic system. Such materials include Z-aminobenzothiazole, pyridine, bipyridyl, bipyridylpyridine, nicotinic acid and the like. Other substances which are non-enzymatic in character but which are eifective in catalyzing the desired oxidation reaction include such compounds as iron sulfocyanate,

iron tannate, iron ferrocyanide, potassium chromic sulfate and others.

In addition to the basic hydrogen peroxide detection system described above, improved color development is spectrophotometer was standardized against distilled water. The results observed were compared with duplicate runs in which the uricase sample contained, in addition to the lyophilized uricase, 0.8 mg. of 6-methoxyobtained if a substance such a polyvinyl alcohol or poly- 5 quinoline. Comparative results are shown in Table I.

TABLE I Absorbance at Uric 620 millimicrons Run Acid, Enzyme Text mg. Solution Percent Without With Additive Additive 8. 0 Uricase Purified e Aqueous. 850 915 8. 0 Uricase Impure b do 700 900 8. 0 Uricase Purified .-do- .790 .885 8. 0 Uricase Impure do .705 925 6.6 Uricase Purified Serum .690 .730 6. 6 Uricase Impure b d 420 685 6. 6 Uricase Purified do 610 .740

' Worthington Purified Uricase No. 5755 (hog liver) is a highly purified enzyme and may be obtained from Worthington Biochemical Corp., Freehold, NJ.

b Worthington Impure Uricase D (hog liver). This sample of uricase was specially prepared by Worthington Biochemical Corp. and is uricase separated at one of the intermediate purification steps prior to obtaining the highly purified enzyme.

0 Laboratory purified uricase (hog described in Biochemica et Biophysica Acta,

liver) was obtained using the purification method vol. 23, 43-53, (1957) d Seravac Uricase No. 4911 (hog liver) is a commercial bulk uricase of low purity and may be obtained from Seravac vinyl pyrrolidone is utilized in conjunction with the chromogenic compound.

As in the case of the enzyme reaction system, the peroxide detecting system may also be utilized in various usable forms. For example, the system may be furnished as a liquid or as a lyophilized powder. If desired, materials in dry form may be mixed and used as a powder or compacted for convenient use in the form of tablets. Another form in which the detecting system may be utilized is as sticks or strips of bibulous materials such as absorbent paper which have been impregnated with the detecting formulation.

The following examples are illustrative of the practice of the present invention and are not to be construed as being any limitation thereto.

EXAMPLE 1 This example demonstrates, in a general way, the improved efiiciency which results when certain substituted quinoline compounds are combined with particular enzymes which are capable of catalyzing .the oxidation of purine based compounds.

A laboratory purified uricase and three grades of commerically purified uricase were utilized in this test. The amount of hydrogen peroxide produced in each of the uricase catalyzed oxidations was deter-mined with a hydrogen peroxide detecting system. The uricase samples comprised 1.20 mg. of lyophilized uricase reconstituted in 0.2 ml. (4 drops) of distilled water. The peroxide detecting system comprised a lyophilized mixture of 0.18 mg. peroxidase and 0.075 mg. of orthotolidine dihydrochloride reconstituted with 0.4 ml.(8 drops) of distilled water. An aqueous test solution containing 8.0 mg. percent of uric acid buttered to a pH of 9.2 with sodium borate and a human blood serum test solution containing 6.6 mg. percent of uric acid were prepared.

To each of the uricase samples, 0.05 ml. (1 drop) of either the aqueous or blood serum test solution was added. The mixtures were then incubated for five minutes at room temperature. Immediately following the five minute incubation period, the peroxide detecting system was combined with each of the incubated mixtures. The amount of peroxide formed was determined colorimetrically by measuring the absorbance of the solution with a spectrophotometer at a wavelength of 620 millimicrons. The

Ltd., Colnbrook, Bucks, England.

EXAMPLE 2 Enzyme reaction system Ml. Sodium borate bufler (0.1 M, pH 9.2) 0.10 Ethylenediaminetetraacetic acid (EDTA) (1% aqueous solution) 0.01 Sodium azide (0.1 M) 0.05 Uricase (aqueous solution 0.02 unit 0.03

e The unit of uricase activity is defined as that quantity of enzyme which will catalyze the conversion of uric acid to allantoin at the rate of one micromole per minute at 25 C. using a uric acid substrate concentration of 20 mg./l. This is referred to by those in the art as Kalckar unit.

The systems so designated in Table II were additionally admixed with 0.8; mg. of the type of substituted quinoline indicated.

One drop (0.05 ml.) of blood serum containing the amount of uric acid indicated in Table II was then added to the enzyme reaction systems prepared above. The reaction mixtures were then incubated for 5 minutes at ambient temperatures after which the following peroxide detectlng system was added to each.

Peroxide detecting system Ml. Sodium succinate buifer (0.5 M, pH 5.1) 0.26 Peroxidase (aqueous solution containing 3 mg./ml.)

(200 units/mg?) 0.05 Orthotolidine dihydrochloride (0.125% aqueous solution) 0.05 Distilled water 0.04

I The unit of peroxidase activity is defined as that quantity of enzyme which will catalyze the decomposition of hydrogen peroxlde at the rate of 1 micromole per 'minute at 25 0., the peroxidase being present in excess.

The absorbance of each system was immediately measured with a spectrophotometer under the conditions indicated in Example 1. The results are recorded in Table II.

TABLE II Urie Acid, Substituted quinoline Absorbance Run mg. percent Enzyme Compound Added at 620 millimiorons 8. 6. 2 Uricase Purified None 610 9 6. 2 Uricase Impure 1L do .360 10.-.- 6.2 do. 6methoxyquinoline .620 11---- 6. 2 do. 2,6-dimethylquinoline 630 12 8. 2 Uricase Purified s. None 820 13 8. 2 Uricase Impure .do 560 14.-.- 8. 2 do. 6-methoxyquinolin 810 s Worthington Purified Uri be obtained from Worthington Biochemical Corp., Freehold, NJ

case No. 5755 (hog liver) is a highly purified enzyme and may Seravac Uricase No. 4911 (hog liver) is an impure bulk enzyme and may be obtained from Seravac Ltd., Colnbrook, Bucks, England.

R2 wherein R and R each independently represent a hydrogen atom or a lower alkyl, lower alkoxy'or formyl group and R represents a hydrogen atom or a lower alkyl, lower alkoxy, carboxyl or formyl group.

What is claimed is:

1. A composition for catalyzing the oxidation of uric acid which comprises an admixture of impure uricase and a substituted quinoline compound represented by the formula:

1'3. wherein R and R each is a member selected from the group consisting of hydrogen, lower alkyl, lower alkoxy and formyl and R is a member selected from the group consisting of lower alkyl, lower alkoxy, carboxyl and formyl.

2. The composition of claim 1 wherein said substituted quinoline compound is 6-methoxyquinoline.

3. The composition of claim 1 wherein said substituted quinoline compound is G-methylquinoline.

4. The composition of claim 1 wherein said substituted quinoline compound is a dimethylquinoline compound.

5. An enzyme system for catalyzing the oxidation of uric acid comprising impure uricase,

a substituted quinoline compound represented by the formula:

wherein R and R each is a member selected from the group consisting of hydrogen, lower alkyl, lower alkoxy and formyl and R is a member selected from the group consisting of lower alkyl, lower alkoxy,

carboxyl and formyl, and

a bufi er effective for maintaining a pH optimal for said oxidation. 6. A composition for the detection of uric acid in blood serum which comprises,

an enzyme reaction system comprising,

impure uricase, a catalase inhibitor, a substituted quinoline compound represented by the formula:

wherein R and R each is a member selected from the group consisting of hydrogen, lower alkyl, lower alkoxy and formyl and R is a member selected from the group consisting of lower alkyl, lower alkoxy, carboxyl and formyl,

a butter for maintaining a pH optimal for said oxidation, and for use in conjunction with and separate from said reaction system,

a peroxide detecting system comprising,

a chromogen,

a substance having peroxidative activity which is effective for catalyzing the oxidation of said chromogen by a peroxide, and

a buffer to maintain a pH which is optimal for chromogen oxidation.

7. A composition according to claim 6 wherein said enzyme reaction system includes a chelating agent capable of preventing heavy metal inhibition of said uricase.

8. The composition of claim 6 wherein said substituted quinoline compound is 6-methoxyquinoline.

9. The composition of claim 6 wherein said substituted quinoline compound is 6-rnethylquinoline.

10. The composition of claim 6 wherein said substituted quinoline compound is a dimethylquinoline compound.

11. The composition of claim 6 wherein said chromogen is orthotolidine.

12. The composition of claim 6 wherein said substance, having peroxidative activity is peroxidase.

13. A composition for the detection of uric acid in blood which comprises,

an enzyme reaction system comprising,

impure uricase,

sodium azide,

ethylenediaminetetraacetic acid,

6-methoxyquinoline, and

a sodium borate buiTer adjusted to a pH of about pH 9.2, and for use in conjunction with and separate from said enzyme reaction system,

a hydrogen peroxide detecting system comprising,

orthotolidine,

peroxidase, and

a sodium succinate buffer adjusted to a pH of about pH 5.1.

14. A process for oxidizing uric acid which comprises adding to said uric acid, in the presence of oxygen, an admixture of impure uricase and a substituted quinoline compound represented by the formula:

Bar-

wherein R and R each is a member selected from the group consisting of hydrogen, lower alkyl, lower alkoxy and formyl and R is a member selected from the group consisting of lower alkyl, lower alkoxy, carboxyl and formyl.

15. In a process for the detection of uric acid in blood serum which comprises adding impure uricase to said blood serum in the presence of oxygen and thereafter measuring the concentration of hydrogen peroxide thereby produced, the improvement which comprises utilizing in admixture with said impure uricase a substituted quinoline compound represented by the formula:

wherein R and R each is a member selected from the group consisting of hydrogen, lower alkyl, lower alkoxy and formyl and R is a member selected from 5 the group consisting of lower alkyl, lower alkoxy, carboxyl and formyl, and a bulfer to maintain an optimal pH for said oxidation.

17. A process for detecting uric acid in blood serum which comprises,

treating said serum with, impure uricase, a catalase inhibitor, a substituted quinoline compound represented by the formula:

it. wherein R and R each is a member selected from the group consisting of hydrogen, lower alkyl, lower alkoxy and formyl and R is a member selected from the group consisting of lower alkyl, lower alkoxy, carboxyl and formyl, and

a bufier to maintain the pH optimal for the oxidation of uric acid, thereby producing a stoichiometric yield of hydrogen peroxide, and

thereafter adding an admixture of,

a substance having peroxidative activity,

a chromogen responsive to said peroxidative activity, and.

a buffer maintaining a pH optimal to said chromogen response and subsequently measuring the chromogenic response produced by the reaction ,of said admixture with said hydrogen peroxide.

18. A process for detecting uric acid in blood serum which comprises,

treating serum with an enzyme reaction system comimpure uricase,

sodium azide,

ethylenediaminetetraacetic acid,

6-methoxyqfuinoline, and

a sodium irrigate buflfer at a pH of 9.2, and

thereafter adding a chromogenically responsive hydrogen peroxide detecting system comprising,

peroxidase,

orthotolidine, and

a sodium succinate buffer at pH 5.1 and subsequently, ineasuring the chromogenic response thereby[ pro duced.

References Cited UNITED STATES PATENTS 3,099,605 7/1963 Free -1035 ALVIN E. TANENHOLTZ, Primary Examiner. 

1. A COMPOSITION FOR CATALYZING THE OXIDATION OF URIC ACID WHICH COMPRISES AN ADMIXTURE OF IMPURE URICASE AND A SUBSTITUTED QUINOLINE COMPOUND REPRESENTED BY THE FORMULA: 